Î²-Catenin Signaling Pathway

Subscriber access provided by Iowa State University | Library

Article

Triazole-Based Inhibitors of the Wnt/#-Catenin Signaling Pathway Improve Glucose and Lipid Metabolism in Diet-Induced Obese Mice Obinna N. Obianom, Yong Ai, yingjun li, Wei Yang, Dong Guo, Hong Yang, Srilatha Sakamuru, Menghang Xia, Fengtian Xue, and Yan Shu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01408 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Triazole-Based Inhibitors of the Wnt/β-Catenin Signaling Pathway Improve Glucose and Lipid Metabolism in Diet-Induced Obese Mice

Obinna N. Obianom,#,1 Yong Ai,#,1 Yingjun Li,1 Wei Yang,≠,1 Dong Guo,1 Hong Yang,1 Srilatha Sakamuru,2 Menghang Xia,2 Fengtian Xue*,1 and Yan Shu*,1,3

1

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,

Baltimore, Maryland 21201, USA 2

National Center for Advancing Translational Sciences, National Institutes of Health,

Bethesda, MD 20892-3375, USA 3

School and Hospital of Stomatology, Guangzhou Medical University, Guangzhou

510140, China #

These authors contributed equally to this work.

≠

Current address: School of Pharmaceutical Engineering, Jiangsu Food & Pharmaceutical

Science College, Huaian, Jiangsu, 223005, China *Correspondence to: Dr. Fengtian Xue at the Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, Maryland 21201, USA; Phone: 410-7068521; Email: [email protected] Dr. Yan Shu at the Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, Maryland 21201, USA; Phone: 410-7067358; Email: [email protected]

1

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Wnt/β-catenin signaling pathway is implicated in the etiology and progression of metabolic disorders. While lines of genetic evidence suggest that blockage of this pathway yields favorable outcomes in treating such ailments, few inhibitors have been used to validate the promising genetic findings. Here, we synthesized and characterized a novel class of triazole-based Wnt/β-catenin signaling inhibitors, and assessed their effects on energy metabolism. One of the top inhibitors, compound 3a, promoted Axin stabilization, which led to the proteasome degradation of β-catenin and subsequent inhibition of the Wnt/βcatenin signaling in cells. Treatment of hepatocytes and high fat diet-fed mice with compound 3a resulted in significantly decreased hepatic lipid accumulation. Moreover, compound 3a improved glucose tolerance of high fat diet-fed mice without noticeable toxicity, while downregulating the genes involved in the glucose and fatty acid anabolism. The new inhibitors are expected to be further developed for the treatment of metabolic disorders.

2


Page 2 of 54

Page 3 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION The Wnt/β-catenin pathway plays a pivotal role in cell proliferation, differentiation and growth.1 It regulates the expression of target genes through the transcriptional factor βcatenin that forms a cytoplasmic “destruction complex” with other proteins including Axin and adenomatous polyposis coli (APC). This complex facilitates the phosphorylation of β-catenin by casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3β), leading to proteasome degradation of β-catenin during the “off state” of the pathway. The “onstate”, on the other hand, involves enhanced stability and accumulation of β-catenin in the cytoplasm, resulting in its increased translocation to the nucleus where it binds to LEF/TCF transcriptional factors and activates the expression of target genes. Wnt/β-catenin pathway crosstalks with many other pathways via key proteins such as β-catenin, Axin, and GSK3β.1,2 Given the crucial function of Wnt/β-catenin pathway, it is not surprising that a strong link between this pathway and metabolic disorders has been recognized in recent years.3-5 The association of the Wnt/β-catenin pathway to metabolic disorders was first established by the identification of a human polymorphism (rs7903146) in the TCF7L2 gene, which encodes a major nuclear partner protein of β-catenin, as a strong risk factor for type-2 diabetes.6

This polymorphism has been known to enhance TCF7L2

transcription.7-9 Later, Savic et al showed that partial knockdown of Tcf7l2 results in metabolic phenotypes of smaller body weights, decreased fasting glucose, and improved glucose tolerance in mice.10 We have published similar observations in mice with the genetic haploinsufficiency of Tcf7l2.11 However, the role of TCF7L2 in maintaining metabolic homeostasis in specific tissues remains controversial. TCF7L2 knockdown was 3



reported to increase glucose production and gluconeogenic gene expression in cultured hepatocytes,12 and the transgenic mice overexpressing a dominant negative Tcf7l2 mutant in the proglucagon gene-expressing cells exhibited defective glucose homeostasis.13 However, Boj et al reported that liver-specific Tcf7l2 knockout led to reduced hepatic glucose production during fasting and improved glucose homeostasis in adult mice on a high-fat diet.14 Recently, Thompson et al reported that liver-specific knockout of β-catenin led to a striking protection from fibrosis and liver injury in mice,15 while Popov et al showed that the hepatic downregulation of β-catenin by anti-sense oligonucleotides could improve insulin sensitivity and glucose tolerance in mice.16 Therefore, although the exact role of the Wnt/β-catenin pathway in metabolism is yet to be illustrated, overall evidence indicates that downregulation of Wnt/β-catenin signaling may provide a new therapeutic strategy for the treatment of metabolic disorders such as fatty liver diseases and diabetes. Therapeutics targeting the Wnt/β-catenin signaling pathway is still in a state of infancy. One reason is that this pathway is bewilderingly complex, with crosstalks to numerous others.17-19 In addition, because of the severe phenotypes observed in genetic knockout animal models,20 safety concern is historically present. Approximately one dozen of Wnt/β-catenin pathway inhibitors with distinct mechanisms have been discovered.21-33 For example, porcupine inhibitors (e.g., LGK-974, Figure 1) decrease the secretion of the Wnt ligands.34 At the plasma membrane, the inhibition of LRP5/6 binding to Wnt proteins by inhibitors such as niclosamide and salinomycin can curb the amount of active βcatenin.22,35 In the cytoplasm, stabilization of the destruction complex (e.g., tankyrase inhibitor XAV93925 and CK1α activator pyrvinium36) can also attenuate β-catenin levels. Of note, the safety concern about Wnt inhibitors has not been borne out either preclinically

4


Page 4 of 54

Page 5 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


or clinically, and recently the β-catenin/CBP inhibitor PRI-724 has entered clinical trials as a potential new treatment of various cancers.17 Several FDA-approved drugs, such as glucocorticoids, retinoids, and celecoxib, are also found to be Wnt/β-catenin pathway inhibitors.17,19 Interestingly, Wnt/β-catenin pathway inhibitors have emerged to replicate the metabolic outcomes of the above-mentioned genetic manipulation in mice. The CK2 inhibitor CX-4945 has been shown to cause mice to be resistant to high fat diet-induced obesity and metabolic disorders.37,38 Treatment of mice with a selective tankyrase inhibitor G007-LK has resulted in profound improvement of glucose tolerance and insulin sensitivity.39 However, further studies are needed to fully establish the applicability of these small molecule inhibitors in the treatment of metabolic disorders. Herein we report a series of novel Wnt/β-catenin pathway inhibitors based upon a triazole scaffold (Figure 1). New compounds were designed by modifying the chemical structure of pyrvinium, an FDA approved anthelmintic effective for pinworm infection. The drug has been reported to inhibit Wnt signaling with a high potency36. However, further development of pyrvinium as a therapeutic agent for metabolic disorders is prohibited by several reasons related to its chemical structure. Pyrvinium possesses a permanently charged N-methylquinolone group that causes extremely low bioavailability of the compound.40 The double bond connecting the two ring systems leads to poor solubility. Moreover, the 2,5-dimethyl-1-phenyl-1H-pyrrole is prone to oxidation41 and known as one of the pan assay interference compounds (PAINs).42 Herein we describe the synthesis of new inhibitors 3a-3u employing a neutral aromatic amino group, an amide linker, and a substituted triazole core. Several of the new compounds showed excellent inhibitory potency against Wnt signaling. One of the new compounds, 3a, was further

5



characterized by various in vitro and in vivo biological assays. Compound 3a showed improved bioavailability, promising efficacy against diet-induced metabolic disorders in mice. In addition, compound 3a was well tolerated in mice without any notable toxicity.

Figure 1. Structures of known Wnt/β-catenin pathway inhibitors LGK974, PRI724, XAV939, pyrvinium, and the design of new triazole-based inhibitors 3a-3u.

RESULTS AND DISCUSSION Synthesis. The synthesis of compounds 3a-3u is detailed in Scheme 1. Diazotization of the amino group of anilines 1a-1n using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) gave azides, which underwent cyclization with β-ketoester in EtONa/EtOH yielded trizaole-3-carboxylic acids 2a-2p. Next, PyCIU-mediated coupling of compounds 2a-2p with various aromatic amines in dichloroethane (DCE) provided target compounds 3a-3r in moderate to good yields. Finally, Pd-catalyzed cross-coupling of compound 3r with various potassium trifluoroborate derivatives yielded products 3s-3u in good yields.

6


Page 6 of 54

Page 7 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Synthesis of compounds 3a-3ua

a

Reagents and conditions: (a) (i) NaNO2, HCl, NaN3, H2O, 0 oC; (ii) ethyl 3-oxobutanoate

for 2a-2n, ethyl 3-oxopentanoate for 2o, ethyl 4-methyl-3-oxopentanoate for 2p, EtONa, EtOH, 80

o

C; (b) 2-aminoquinoline for 3a-3p, naphthalen-2-amine for 3q, 6-

bromoquinolin-2-amine for 3r, PyCIU, DIPEA, DCE, 80 oC; (c) potassium trifluoroborate derivatives, Pd(OAc)2, XPhos, Cs2CO3, THF/H2O, 80 oC, 24-48 h.

7



Structure-Activity Relationship. Compound 3a indicated an excellent IC50 value of 4.1 nM in the luciferase gene reporter assay for Wnt signaling activity, which is over 1,000fold higher than that of 3b (3-methyl group) and 3c (4-methyl group), and 180-fold higher than the parent compound pyrvinium (Table 1). Additional methyl group at the 3- (3l) or 4-position (3m) of the phenyl ring led to new inhibitors with 34- and 66-fold decreased potencies, respectively. The naphthyl analog 3n was a weak inhibitor with an IC50 value of 5.5 µM. These results indicated that single ortho-substitution is preferred on the phenyl ring. Substitution of the 2-methyl group with various functionalities generated new inhibitors 3d-3k. Among them, the F-analog (3d) demonstrated excellent potency with an IC50 value in the sub-nanomolar range. The Br- (3e), NC- (3f), and MeO- (3g) analogs also showed low nM potencies. However, large substituents such as amide (3h), ketone (3i), morpholine (3j) and Ph (3k) turned out to be detrimental to the inhibitory activity. Next, substitution effects of the triazole core (R2) were studied using compounds 3o and 3p. Compared to inhibitor 3d, the ethyl analog 3o was slightly less potent. However with the branched isopropyl group, compound 3p turned out to be over 20-fold less potent than compound 3d. These results indicated that small group was preferred at the R2 position. In addition, removal of the quinoline nitrogen yielded compound 3q that totally lost inhibitory activity. This result highlighted the importance of the quinoline nitrogen in maintaining high potency. Finally, we sought to explore the possibility of expanding the quinoline ring system. The Br-substituted compound 3r indicated similar potency as that of the parent compound pyrvinium. Similarly, potent inhibitors were obtained when morpholine (3s), piperidine (3t), or thiomorpholine (3u) were included at the same R3 8


Page 8 of 54

Page 9 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


position. These results indicated that further expansion of the quinoline ring could impair the activity of the inhibitor. Table 1. Inhibition of Wnt/β-Catenin Signaling Pathway by Compounds 3a-3u

Cmpds

R1

R2

R3

X

IC50 (nM)a

3a

2-Me

Me

H

N

4.1 ± 0.4

3b

3-Me

Me

H

N

>10,000

3c

4-Me

Me

H

N

>10,000

3d

2-F

Me

H

N

1.2 ± 0.2

3e

2-Br

Me

H

N

7.6 ± 0.3

3f

2-CN

Me

H

N

34 ± 4.3

3g

2-OMe

Me

H

N

18 ± 3.3

3h

2-CONH2

Me

H

N

>10,000

3i

2-COMe

Me

H

N

8,800 ± 1322

3j

2-morpholine

Me

H

N

1,900 ± 5.4

3k

2-Ph

Me

H

N

390 ± 68.1

3l

2,3-diMe

Me

H

N

140 ± 5.8

3m

2,4-diMe

Me

H

N

270 ± 23.8

3n

2,3-Ph (fused)

Me

H

N

5,500 ± 1186

3o

2-F

Et

H

N

4.7 ± 0.7

3p

2-F

i-Pr

H

N

25 ± 0.5

3q

2-Me

Me

H

CH

4,900 ± 743

3r

2-Me

Me

Br

N

830 ± 98.2

3s

2-Me

Me

N

1,300 ± 169

3t

2-Me

Me

N

1,200 ± 168

9



3u

2-Me

Me

pyrvinium a

Page 10 of 54

N

230 ± 28 750 ± 137

The values of IC50 for each compound to inhibit the Wnt signaling activity, as determined

from the luciferase reporter gene assay, were calculated and data are expressed as mean IC50 (nM) ± SE of each compound from three independent experiments. Note that compounds 3a-3u did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay (Table S2). Compound 3a, a Potent Inhibitor of the Wnt/β-Catenin Signaling Pathway. As model compounds, we next chose compounds 3a and 3n and further evaluated them in various biological assays. We chose compound 3a over the more potent compound 3d (Table 1) because in the assessment of their potential cytotoxicity in unstimulated normal HEK293 cells with a longer incubation time of 72 h relative to that for the above reporter assay, we found that 3d was more cytotoxic than 3a (Figure S1). While compound 3d was more potent than 3a, their potencies remained in the same magnitude. Compound 3a showed superior potency in HEK293 cells in the presence of Wnt signaling activators LiCl and Wnt3a (Figure 2A, B). Compound 3a inhibited the Wnt/β-catenin signaling pathway by stabilization of Axin and subsequent β-catenin degradation (Figure 2C-E and Figure S2AB). Stabilization of Axin was confirmed by the increased cytoplasmic punctas as has been demonstrated previously by other Axin stabilizers.43 The expression of the pathway target genes including CycD1 and Axin2 was repressed at the messenger RNA (mRNA) levels. In the presence of LiCl, which activates Wnt/β-catenin pathway via the inhibition of GSK3β, compound 3a decreased the cellular level of β-catenin while the inactive analog 3n had little effect (Figure 2F). 10


Page 11 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The downregulation of β-catenin levels and the consistent efficacy of compound 3a in both Wnt3a- and LiCl-conditioned medium suggested a mechanism different from that of the parent compound pyrvinium, whose effect is diminished in the presence of LiCl.36 We performed additional studies with varying concentrations of LiCl, and observed no effect by LiCl in changing the inhibitory potency of compound 3a towards Wnt/β-catenin signaling pathway (Figure S2).

Figure 2. Characterization of compound 3a as an inhibitor of Wnt/β-catenin signaling pathway. (A) TCF/LEF responsive luciferase reporter assay in HEK293 cells with varying concentration of compound 3a and (B) compound 3n in Wnt3a- or LiCl-conditioned medium. The data were fitted to determine the IC50 of inhibition of LiCl- and Wnt3ainduced activation of the Wnt/β-catenin signaling pathway (mean ± S.D, n = 4). (C) Confocal microscope images of 3a-treated HEK293 cells for detection of Axin (Axin1). The cells were incubated with 1 µM of compound 3a for 14 h. The nucleus was stained by DAPI. (D) Western blot analysis showing the effect of compound 3a treatment on the 11



Page 12 of 54

levels of β-catenin and Axin1 proteins in HEK293 cells in the presence of WNT3a. The cells were treated with the compound for 2 h. (E) The mRNA expression of the target genes of Wnt/β-catenin signaling pathway. The cells were treated for 48 h and harvested with TriZol reagent. *p < 0.05. (F) Western blot analysis of HEK293 cells treated with compounds 3a and 3n in the presence of lithium chloride for 24 h. RLU- Relative light units. Mechanism of Wnt/β-catenin Pathway Inhibition by Compound 3a.

Sequential

phosphorylation of β-catenin on residues Ser33, Ser37 and Thr41 by GSK3β is a critical step in β-catenin degradation. By causing increase in the phosphorylation at these sites, many small molecule Wnt inhibitors facilitate the degradation of β-catenin. To further ascertain if our new inhibitors function through a similar mechanism, we examined the inhibitory potency of compound 3a in HEK293 cells overexpressed with wild-type and mutant (S33Y) β-catenin (Figure S3).

Overexpression of both exogenous β-catenin

plasmids led to significant increases in Wnt signaling activities as reflected by the luciferase reporter gene assay, with the S33Y mutant giving a larger increase than the wild type.

Nonetheless, compound 3a inhibited Wnt signaling with a similar potency

irrespective of overexpression of either β-catenin, suggesting that the inhibitory effects of compound 3a may be independent of GSK3β phosphorylation of at least Ser33 on βcatenin. Note that aside from Ser33 phosphorylation, GSK3β also phosphorylates Ser37 and Thr41 of β-catenin. Thus the mutated β-catenin construct used in our assay may not be sufficient to impair the function of GSK3β, as immunoprecipitation of β-catenin showed that compound 3a strengthened the binding of GSK3β and Axin to β-catenin (Figure 3A).

12


Page 13 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thus, the results suggest that compound 3a treatment may lead to fortification of the destruction complex to propagate the degradation of β-catenin. We next silenced major components of the β-catenin destruction complex, CK1α, GSK3β and Axin1, to assess their contribution to the effect of inhibitor 3a. Only the knockdown of CK1 and GSK3β partially abolished the Wnt inhibitory effect while Axin1 silencing had no effect on the gene reporter assay of inhibitor 3a (Figure 3B). Next, we performed surface plasmon resonance (SPR) analysis using recombinant CK1α, GSK3β, and Axin to determine the binding affinity of compound 3a to these proteins. We found that compound 3a could not bind to CK1α and Axin (data not shown), while exhibiting a weak binding (KD = 4.3 µM) for recombinant GST-tagged GSK3β (Figure S4A-C). Further, we knocked down GSK3β and performed the reporter assay for compound 3a. The results show that exclusion of GSK3β only decreased the effect of compound 3a by 1.7 fold (IC50 of si-control = 20 nM vs. si-GSK3β = 34 nM) (Figure S4D). The noncorrelation of the IC50 value from the reporter assay to the KD of weak binding to GSK3β suggests that GSK3β is probably not the main target of compound 3a, although it may play a direct role in the inhibitory effect of compound 3a on Wnt/β-catenin signaling pathway. In order to determine the inhibitory effect of compound 3a in other cells bearing mutations that lead to an activated Wnt/β-catenin pathway, we overexpressed the luciferase reporter constructs in SW480 and HepG2 cell lines. The SW480 cells have inactivating mutations in APC, which lead to ineffective tethering of β-catenin to the destruction complex and subsequent increase in cytoplasmic and nuclear β-catenin levels.44

In

contrast, HepG2 cells contain a deletion of the amino acids 25-140 of the CTNNB1 (encoding β-catenin) gene, which includes the binding sites of GSK3β and CK1α (Figure 13



3C).45 Inhibition of the reporter gene activity by compound 3a was well observed in SW480 cells, but insignificant in HepG2 cells (Figure 3D). These results suggest the necessity of the full length of β-catenin with intact GSK3β and CK1α sites for the inhibitory activity of compound 3a.

Figure 3. Role of Wnt/β-catenin pathway effector proteins in the inhibition by compound 3a. (A) Immunoprecipitation of β-catenin in HEK293 to determine the effect of compound 3a treatment (12 h) on the interaction of β-catenin with other pathway effector proteins. (B) TCF/LEF gene reporter assay of compound 3a in the presence of knockdown of GSK3β, Axin and CK1α. The HEK293 cells were treated with or without compound 3a (20 nM) for 24 h. (C) Western blot analysis of protein expression of β-catenin in SW480 and HepG2 cells. (D) TCF/LEF gene reporter assay of SW480 and HepG2 cells treated

14


Page 14 of 54

Page 15 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with compound 3a. Data are represented as mean ± S.D, n = 3. RLU- relative light units. *** p < 0.001. Compound 3a Decreased Lipid Accumulation and Altered the Expression of Lipogenic and Gluconeogenic Genes in Hepatocytes. It has been reported that the Wnt/β-catenin pathway may play a role in cellular metabolism. Specifically, Axin has been implicated in lipid metabolism. In mice, knockdown of Axin results in significantly increased hepatic lipid accumulation.46 Because compound 3a stabilized the cellular level of Axin, we performed a Nile red staining assay to examine the effect of compound 3a on lipid accumulation in human hepatic Huh7 cells (Figure 4A). Treatment of the cells with compound 3a dramatically decreased lipid accumulation. Similar effects were observed by a known Axin stabilizer XAV939. Next, we performed a quantitative PCR to determine the effect of compound 3a on the gene expression of lipogenesis and gluconeogenesis in mouse hepatocytes. Our results showed that compound 3a downregulated the mRNA levels of the gluconeogenic (PEPCK and G6PASE) and lipogenic genes (FASN, ACAA1A, ACOT4, and SCD1) (Figure 4B). These results suggested that compound 3a might decrease the accumulation of lipids in hepatocytes by downregulating gluconeogenic and lipogenic pathways as a consequence of Axin stabilization.

15



Figure 4. Compound 3a decreased lipid accumulation and the expression of lipogenic and gluconeogenic genes in hepatocytes. (A) Nile red staining assay of the Huh7 cells treated with 5 µM of compounds XAV939 or 3a for 36 h and together with 200 µM oleate for 16 h. TD, transmitted light differential interference contrast image. (B) The mRNA expression of various lipogenic and gluconeogenic genes in normal mouse hepatocytes treated with 5 µM of compound 3a. Data represents mean ± S.D. of triplicates. In Vivo Efficacy of Compound 3a against Diet-Induced Metabolic Disorders in Mice. With the promising in vitro effects of compound 3a on metabolism in hepatocytes, we

16


Page 16 of 54

Page 17 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


further studied its efficacy in vivo against metabolic disorders using a mouse model. Initially, we briefly assessed the pharmacokinetic and physicochemical properties of compound 3a. Compared to the reported parameters for pyrvinium, our new analogue showed improved physicochemical and pharmacokinetic properties with an oral bioavailability of 21% (pyrvinium has less than 1%). The plasma half-life of compound 3a is 2.8 – 3.3 h, with an oral maximal concentration of 2.2 µg/mL in the plasma after a single dose of 10 mg/kg (Table S3). We then proceeded to efficacy studies in the high fat diet-fed mouse model. We performed a pilot dose escalation study to determine the optimal dose for compound 3a and to ensure little or no toxicity to the mice (data not shown). While we did not observe any noticeable toxicity at up to 200 mg/kg of compound 3a, we found promising efficacy and hepatic improvement at 40 mg/kg so we chose this dose to conduct a more comprehensive study in mice. Wild type C57BL/6J mice were fed with a high fat diet or normal chow diet for 6 weeks before treatment. The mice were then divided into four groups: two were fed normal chow and the other two were fed with the high fat diet. Compound 3a was administered intraperitoneally every 2 days at 40 mg/kg for 11 weeks, a dose selected based on the pilot dose escalation studies. After 11 weeks of treatment, compound 3a significantly improved glucose tolerance in the high fat diet group (Figure 5A-B). The inhibition of Wnt signaling and the improvement of glucose tolerance by compound 3a were confirmed by the decreased hepatic mRNA levels of Wnt target genes and those of gluconeogenesis and lipogenesis in the mice fed with the high fat diet, respectively (Figure 6A). Of note, the effects of compound 3a on gene expression were insignificant in the mice fed with the normal chow diet, suggesting a selectivity of compound 3a towards metabolic disorders (Figure 6B).

17



Figure 5. Improvement of glucose tolerance by compound 3a in C57BL/6J mice fed with a high fat diet. (A) Intraperitoneal glucose tolerance test (IPGTT) was carried out on the mice fed with the high fat diet (HFD) and normal chow diet (NC) at the start of treatment (WK0) and after 11 weeks (WK11) of treatment. The mice received intraperitoneal injection of 40 mg/kg compound 3a or vehicle (corn oil) every two days. (B) The area under the curve (AUC, min*mg/dL) for the IPGTT. Data represents mean ± SEM, n = 5 per group. *p < 0.05 as compared to the vehicle group.

18


Page 18 of 54

Page 19 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Effects of compound 3a on the hepatic expression of select Wnt/β-catenin pathway target genes and those involved in energy metabolism in mice. (A) The mRNA expression of select genes in the mice fed with a high fat diet. (B) The mRNA expression of select genes in the mice fed with normal chow diet. The mice received i.p. injection of

19



40 mg/kg compound 3a or vehicle (corn oil) every two days for 11 weeks. Data represents mean ± SEM, n = 5 per group. * p < 0.05, ** p < 0.01. To further assess the efficacy of compound 3a against the metabolic disorders induced by the high fat diet in mice, we conducted additional measurements. The body weight and the liver weight adjusted by the body weight were increased in the high fat diet-fed mice. However, the increases were suppressed by the treatment of compound 3a. Of note, the treatment caused no effects in the normal chow diet-fed mice (Figure 7). Consistently, in our hepatic histological examination, compound 3a treatment drastically reduced the hepatic lipid accumulation induced by the high fat diet. In addition, the hepatic triglyceride content and the serum cholesterol level were reduced by the treatment of compound 3a in the high fat diet-fed mice. Importantly, the mice that received compound 3a treatment did not exhibit any significant toxicity as indicated by the blood chemical measurements of liver and kidney function and by the histological examination (Table 2, Figure S5). Taken together, the effects of compound 3a were pronounced in the mice with metabolic disorders induced by the high fat diet. Inhibition of the Wnt signaling pathway appeared to mediate the efficacy of compound 3a against the metabolic disorders induced by the high fat diet because the results phenocopied those seen in genetic knockdown of Axin, Ctnnb1 (βcatenin) and Tcf7l2 in the mice fed a high fat diet.11,46,47

20


Page 20 of 54

Page 21 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Effects of compound 3a treatment on body weight gain and hepatic lipid accumulation in C57BL/6J mice. (A) Body weight gain in high fat diet and normal chow group mice treated with 40 mg/kg compound 3a or vehicle (corn oil). (B) Hematoxylin and eosin staining for liver tissue samples. (C) Hepatic triglyceride content and (D) the ratio of liver/body weight in high fat diet-fed mice. The mice received i.p. injection of 40 mg/kg compound 3a or vehicle (corn oil) every two days for 11 weeks. V= vehicle, 3a = Compound 3a. * p < 0.05.

21



Page 22 of 54

Table 2. Serum Biochemical Parameters in Mice Received Compound 3a or Vehicle High fat diet (n=10)

Physiologic

Normal chow (n=10)

Unit parameter

vehicle

3a

p-value

vehicle

3a

p-value

ALT

U/L

63.3 ± 26.9

21.0 ± 1.9

NS

20.0 ± 5.1

12.8 ± 1.6

NS

Alkaline

U/L

58.3 ± 3.8

51.0 ± 4.6

NS

66.5 ± 1.8

57.5 ± 1.1

NS

AST

U/L

131 ± 15.6

94.8 ± 17.2

NS

119 ± 28.3

74.5 ± 6.25

NS

Total bilirubin

mg/dL

0.20 ± 0.0

0.20 ± 0.03

NS

0.20 ± 0.0

0.20 ± 0.0

NS

Cholesterol

mg/dL

316 ± 25.0

223 ± 18.1

0.003

183 ± 16.6

194 ± 13.7

NS

Creatinine Jaffe

mg/dL

0.30 ± 0.02

0.20 ± 0.02

NS

0.20 ± 0.01

0.2 ± 0.01

NS

LDH

U/L

469 ± 67

313 ± 40.8

Î²-Catenin Signaling Pathway

Recommend Documents